Differential phosphorylation of two serine clusters in mouse HORMAD1 during meiotic prophase I progression.

Mouse HORMAD1 is a phospho-protein involved in multiple functions during meiotic prophase I. To obtain insight into the significance of its phosphorylation, we generated phospho-specific antibodies against two serine residues, Ser307 and Ser378, representing each of two serine clusters in mouse HORMAD1. The Ser307 phosphorylation is detectable from early leptotene substage in both wild-type and Spo11-/- spermatocytes, indicating that Ser307 is a primary and SPO11-independent phosphorylation site. In contrast, the Ser378 phosphorylation is negligible at earlier substages in wild-type and Spo11-/- spermatocytes. After mid-zygotene substage, the Ser378 phosphorylation is abundant on unsynapsed chromosome axes in wild-type spermatocytes and is detected only in a part of unsynapsed chromosome axes in Spo11-/- spermatocytes. We also generated a non-phosphorylated Ser307-specific antibody and found that Ser307 is phosphorylated on sex chromosome axes but is almost entirely unphosphorylated on desynapsed chromosome axes in diplotene spermatocytes. These results demonstrated a substage-specific phosphorylation status of mouse HORMAD1, which might be associated with multiple substage-specific functions.

Aquaporin-5 Protein Is Selectively Reduced in Rat Parotid Glands under Conditions of Fasting or a Liquid Diet.

Aquaporin-5 (AQP5) water channel, transmembrane protein 16A (TMEM16A) Ca2+-activated Cl- channel, and Na+-K+-2Cl- cotransporter (NKCC1) are membrane proteins on salivary gland acinar cells that function in watery saliva secretion. We examined their expression changes in rat parotid glands under reduced mastication. Rats were either fed regular chow as a control group, fasted for 48 hr or fed a liquid diet for 48 hr or 1 week to reduce mastication. The parotid glands were then resected to analyze the protein and mRNA levels by immunofluorescence, immunoblotting, and reverse-transcription quantitative PCR (RT-qPCR). AQP5 protein was significantly decreased in both liquid diet groups and the fasting group but its mRNA levels showed no apparent changes compared with the control group. The protein and mRNA levels of TMEM16A and NKCC1 showed no significant changes between any of the groups other than an increase in NKCC1 mRNA in the 1-week liquid diet group. These results suggest that reduced mastication may increase the AQP5 protein degradation, but not that of other membrane proteins necessary for saliva secretion.

TRPC5 regulates axonal outgrowth in developing retinal ganglion cells.

The TRPC5 ion channel is activated upon depletion of intracellular calcium stores, as well as by various stimuli such as nitric oxide (NO), membrane stretch, and cold temperatures. TRPC5 is abundantly expressed in the central nervous system where it has important neuronal functions. In the chick retina, TRPC5 expression was shown to be restricted to amacrine cells (ACs) and Müller glial cells, although its expression was also observed in the ganglion cell layer (GCL) in displaced ACs, as determined by their characteristic cell morphology. However, it is possible that this expression analysis alone might be insufficient to fully understand the expression of TRPC5 in retinal ganglion cells (RGCs). Hence, we analyzed TRPC5 expression by in situ hybridization and immunostaining in the developing mouse retina, and for the first time identified that developing and mature RGCs strongly express TRPC5. The expression begins at E14.5, and is restricted to ACs and RGCs. It was reported that TRPC5 negatively regulates axonal outgrowth in hippocampal neurons. We thus hypothesized that TRPC5 might have similar functions in RGCs since they extend very long axons toward the brain, and this characteristic significantly differs from other retinal cell types. To elucidate its possible involvement in axonal outgrowth, we inhibited TRPC5 activity in developing RGCs which significantly increased RGC axon length. In contrast, overexpression of TRPC5 inhibited axonal outgrowth in developing RGCs. These results indicate that TRPC5 is an important negative regulator of RGC axonal outgrowth. Since TRPC5 is a mechanosensor, it might function to sense abnormal intraocular pressure changes, and could contribute to the death of RGCs in diseases such as glaucoma. In this case, excessive Ca2+ entry through TRPC5 might induce dendritic and axonal remodeling, which could lead to cell death, as our findings clearly indicate that TRPC5 is an important regulator of neurite remodeling.

A case of severe headache attributed to vertebral artery dissection.

BACKGROUND: Vertebral artery dissection (VAD) sometimes has no specific symptoms and is difficult to differentiate from other forms of headache. CASE PRESENTATION: A woman in her thirties had a severe, throbbing left-sided headache. A migraine without aura was suspected and zolmitriptan was administered, which alleviated the symptoms. The woman was consequently deemed to have a migraine without aura. Despite the lack of abnormal neurological findings and showed no abnormalities on cranial computed tomography, her symptoms were not typical for migraines and showed little improvement with therapy. She therefore underwent a cranial magnetic resonance imaging (MRI) examination, which revealed VAD, for which she was transferred to the department of neurosurgery for conservative treatment. CONCLUSION: The possibility of vertebral artery dissection should be considered in the differential diagnosis of severe secondary headaches, and prompt diagnosis and treatment based on detailed MRI and magnetic resonance angiography examinations should be performed.

BMP4 signaling in NPCs upregulates Bcl-xL to promote their survival in the presence of FGF-2.

We previously reported that BMP4 does not promote proliferation or differentiation of CD44-positive astrocyte precursor cells (APCs) but greatly promotes their survival in the presence of fibroblast growth factor-2 (FGF-2). In this study, we examined if BMP4 acts as a survival factor also for neural stem/progenitor cells (NPCs) isolated from ganglionic eminence of neonatal mouse brain. We found BMP4 promotes survival but not proliferation or differentiation of these cells, just as in the case for CD44-positive APCs. Microarray analysis revealed some candidate molecules in the signaling pathway downstream of BMP4. Among them, we focused on Id1 (inhibitor of DNA-binding 1) and Bcl-xL in this study. Expression of both genes was promoted in the presence of BMP4, and this promotion was reduced by dorsomorphin, an inhibitor of BMP4 signaling. Furthermore, cytochrome c release from mitochondria was significantly reduced in the presence of BMP4, suggesting up-regulation of Bcl-xL activity by BMP4. Id1 siRNA reduced the expression of Bcl-xL, and negated survival promoting effect of BMP4. These data suggest that BMP4 promotes survival of NPCs by enhancing the anti-apoptotic function of Bcl-xL via BMP4-Smad1/5/8-Id1 signaling.

Fibronectin on extracellular vesicles from microvascular endothelial cells is involved in the vesicle uptake into oligodendrocyte precursor cells.

We previously reported transplantation of brain microvascular endothelial cells (MVECs) into cerebral white matter infarction model improved the animal's behavioral outcome by increasing the number of oligodendrocyte precursor cells (OPCs). We also revealed extracellular vesicles (EVs) derived from MVECs promoted survival and proliferation of OPCs in vitro. In this study, we investigated the mechanism how EVs derived from MVECs contribute to OPC survival and proliferation. Protein mass spectrometry and enzyme-linked immunosorbent assay revealed fibronectin was abundant on the surface of EVs from MVECs. As fibronectin has been reported to promote OPC survival and proliferation via integrin signaling pathway, we blocked the binding between fibronectin and integrins using RGD sequence mimics. Blocking the binding, however, did not attenuate the survival and proliferation promoting effect of EVs on OPCs. Flow cytometric and imaging analyses revealed fibronectin on EVs mediates their internalization into OPCs by its binding to heparan sulfate proteoglycan on OPCs. OPC survival and proliferation promoted by EVs were attenuated by blocking the internalization of EVs into OPCs. These lines of evidence suggest that fibronectin on EVs mediates their internalization into OPCs, and the cargo of EVs promotes survival and proliferation of OPCs, independent of integrin signaling pathway.

Monitored anesthesia care based on ultrasound-guided subcostal transversus abdominis plane block for continuous ambulatory peritoneal dialysis catheter surgery: case series.

Planning safe perioperative management for patients undergoing continuous ambulatory peritoneal dialysis (CAPD) catheter surgery (insertion and extraction of the catheter) is often difficult because many of these patients not only have renal insufficiency but also have co-existing disorders, such as heart diseases. As increased indications for perioperative anticoagulation therapy have limited the choice of anesthesia, selecting an appropriate anesthetic method, particularly for patients with poor systemic conditions, is becoming more challenging. We report seven cases of CAPD catheter surgery successfully managed by monitored anesthesia care using subcostal transversus abdominis plane (TAP) block with additional local anesthetic infiltration and analgesics. Despite co-existing cardiac disease and/or coagulation disorders, all patients were safely managed without any other major anesthetic methods. Subcostal TAP block is a useful anesthetic option for CAPD catheter surgery, particularly for patients with poor systemic conditions and/or in whom neuraxial blocks are contraindicated.

1α,25-dihydroxyvitamin D3 modulates the hair-inductive capacity of dermal papilla cells: therapeutic potential for hair regeneration.

Dermal papilla cells (DPCs) have the potential to induce differentiation of epithelial stem cells into hair, and Wnt signaling is deeply involved in the initiation process. The functional limitation of expanded adult DPCs has been a difficult challenge for cell-based hair regrowth therapy. We previously reported that 1α,25-dihydroxyvitamin D(3) (VD(3)) upregulates expression of transforming growth factor (TGF)-ß2 and alkaline phosphatase (ALP) activity, both features of hair-inducing human DPCs (hDPCs). In this study, we further examined the effects and signaling pathways associated with VD(3) actions on DPCs. VD(3) suppressed hDPC proliferation in a dose-dependent, noncytotoxic manner. Among the Wnt-related genes investigated, Wnt10b expression was significantly upregulated by VD(3) in hDPCs. Wnt10b upregulation, as well as upregulation of ALPL (ALP, liver/bone/kidney) and TGF-ß2, by VD(3) was specific in hDPCs and not detected in human dermal fibroblasts. Screening of paracrine or endocrine factors in the skin indicated that all-trans retinoic acid (atRA) upregulated Wnt10b gene expression, although synergistic upregulation (combined atRA and VD(3)) was not seen. RNA interference with vitamin D receptor (VDR) revealed that VD(3) upregulation of Wnt10b, ALPL, and TGF-ß2 was mediated through the genomic VDR pathway. In a rat model of de novo hair regeneration by murine DPC transplantation, pretreatment with VD(3) significantly enhanced hair folliculogenesis. Specifically, a greater number of outgrowing hair shafts and higher maturation of regenerated follicles were observed. Together, these data suggest that VD(3) may promote functional differentiation of DPCs and be useful in preserving the hair follicle-inductive capacity of cultured DPCs for hair regeneration therapies.

Cdc6 protein activates p27KIP1-bound Cdk2 protein only after the bound p27 protein undergoes C-terminal phosphorylation.

In mammalian cells Cdk2 activity during the G(1)-S transition is mainly controlled by p27(KIP1). Although the amount and subcellular localization of p27 influence Cdk2 activity, how Cdk2 activity is regulated during this phase transition still remains virtually unknown. Here we report an entirely new mechanism for this regulation. Cdc6 the AAA+ ATPase, known to assemble prereplicative complexes on chromosomal replication origins and activate p21(CIP1)-bound Cdk2, also activated p27-bound Cdk2 in its ATPase and cyclin binding motif-dependent manner but only after the p27 bound to the Cdk2 was phosphorylated at the C terminus. ROCK, which mediates a signal for cell anchorage to the extracellular matrix and activates the mTORC1 cascade as well as controls cytoskeleton assembly, was partly responsible for C-terminal phosphorylation of the p27. In vitro reconstitution demonstrated ROCK (Rho-associated kinase)-mediated phosphorylation of Cdk2-bound p27 at the C terminus and subsequent activation of the Cdk2 by Cdc6.

Mammalian target of rapamycin complex 1 signaling opposes the effects of anchorage loss, leading to activation of Cdk4 and Cdc6 stabilization.

When deprived of an anchorage to the extracellular matrix, fibroblasts arrest in the G(1) phase with inactivation of Cdk4/6 and Cdk2 and destruction of Cdc6, the assembler of prereplicative complexes essential for S phase onset. How cellular anchorages control these kinases and Cdc6 stability is poorly understood. Here, we report that in rat embryonic fibroblasts, activation of mammalian target of rapamycin complex 1 by a Tsc2 mutation or overexpression of a constitutively active mutant Rheb overrides the absence of the anchorage and stabilizes Cdc6 at least partly via activating Cdk4/6 that induces Emi1, an APC/C(Cdh1) ubiquitin ligase inhibitor.

Cdc6 determines utilization of p21(WAF1/CIP1)-dependent damage checkpoint in S phase cells.

When cells traversing G(1) are irradiated with UV light, two parallel damage checkpoint pathways are activated: Chk1-Cdc25A and p53-p21(WAF1/CIP1), both targeting Cdk2, but the latter inducing a long lasting arrest. In similarly treated S phase-progressing cells, however, only the Cdc25A-dependent checkpoint is active. We have recently found that the p21-dependent checkpoint can be activated and induce a prolonged arrest if S phase cells are damaged with a base-modifying agent, such as methyl methanesulfonate (MMS) and cisplatin. But the mechanistic basis for the differential activation of the p21-dependent checkpoint by different DNA damaging agents is not understood. Here we report that treatment of S phase cells with MMS but not a comparable dose of UV light elicits proteasome-mediated degradation of Cdc6, the assembler of pre-replicative complexes, which allows induced p21 to bind Cdk2, thereby extending inactivation of Cdk2 and S phase arrest. Consistently, enforced expression of Cdc6 largely eliminates the prolonged S phase arrest and Cdk2 inactivation induced with MMS, whereas RNA interference-mediated Cdc6 knockdown not only prolongs such arrest and inactivation but also effectively activates the p21-dependent checkpoint in the UV-irradiated S phase cells.

ATP-dependent activation of p21WAF1/CIP1-associated Cdk2 by Cdc6.

When cells progressing in mid-S phase are damaged with a base-modifying chemical, they arrest in S phase long after the CHK1 checkpoint signal fades out, partly because of p53-mediated long-lasting induction of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1). We have recently found that enforced expression of Cdc6, the assembler of prereplicative complexes, markedly advances recovery from the prolonged S-phase arrest and reactivation of Cdk2 despite the presence of a high level of induced p21. Here, we report that Cdc6 protein can activate p21-associated Cdk2 in an ATP-dependent manner in vitro. Consistently, Cdc6 mutated for ATPase or a putative cyclin binding motif is no longer able to activate the Cdk2 in vitro or promote reinitiation of S-phase progression and reactivation of Cdk2 in vivo. These results reveal the never anticipated function of Cdc6 and redefine its role in the control of S-phase progression in mammalian cells.

Chemical DNA damage activates p21 WAF1/CIP1-dependent intra-S checkpoint.

When cells progressing in G(1) phase are irradiated with UV light, two damage checkpoint pathways are activated: CHK1-Cdc25A and p53-p21WAF1/CIP1, both targeting Cdk2 but the latter inducing long lasting inactivation. In similarly irradiated S phase cells, however, p21WAF1/CIP1-dependent checkpoint is largely inactive. We report here that p21-dependent checkpoint can effectively be activated and induce a prolonged S phase arrest with similarly extended inactivation of Cdk2 by association of p21 if mid-S phase cells are damaged with a base-modifying agent instead of UV light, indicating that the poor utilization of p21-dependent checkpoint is not an innate property of S phase cells.

Recapitulation of in vivo gene expression during hepatic differentiation from murine embryonic stem cells.

Hepatic differentiation at the molecular level is poorly understood, mainly because of the lack of a suitable model. Recently, using adherent monoculture conditions, we demonstrated the direct differentiation of hepatocytes from embryonic stem (ES) cells. In this study, we exploited the direct differentiation model to compare the gene expression profiles of ES cell-derived hepatocytes with adult mouse liver using DNA microarray technology. The results showed that the ES cell-derived hepatocyte gene expression pattern is very similar to adult mouse liver. Through further analysis of gene ontology categories for the 232 most radically altered genes, we found that the significant categories related to hepatic function. Furthermore, through the use of small interfering RNA technology in vitro, hepatocyte nuclear factor 3beta/FoxA2 was identified as having an essential role in hepatic differentiation. These results demonstrate that ES cell-derived hepatocytes recapitulate the gene expression profile of adult mouse liver to a significant degree and indicate that our direct induction system progresses via endoderm differentiation. In conclusion, our system closely mimics in vivo hepatic differentiation at the transcriptional level and could, therefore, be useful for studying the molecular basis of hepatocyte differentiation per se.

Direct hepatic fate specification from mouse embryonic stem cells.

The molecules responsible for hepatic differentiation from embryonic stem (ES) cells have yet to be elucidated. Here we have identified growth factors that allow direct hepatic fate-specification from ES cells by using simple adherent monolayer culture conditions. ES cell-derived hepatocytes showed liver-specific characteristics, including several metabolic activities, suggesting that ES cells can differentiate into functional hepatocytes without the requirement for embryoid body (EB) formation, in vivo transplantation, or a coculture system. Most importantly, transplantation of ES cell-derived hepatocytes in mice with cirrhosis showed significant therapeutic effects. In conclusion, this novel system for hepatic fate specification will help elucidate the precise molecular mechanisms of hepatic differentiation in vitro and could represent an attractive approach for developing stem cell therapies for treatment of hepatic disease in humans. Supplementary material for this article can be found on the HEPATOLOGY website ( http://www.interscience.wiley.com/jpages/0270-9139/suppmat/index.html).

HST-1/FGF-4 plays a critical role in crypt cell survival and facilitates epithelial cell restitution and proliferation.

The fibroblast growth factor-4 (HST-1/FGF-4) is a heparin-binding growth factor that influences on epithelial and many other cells through interaction with FGF receptors. It has been demonstrated that the HST-1/FGF-4 gene protects mice from lethal irradiation by preventing bone marrow damage and intestinal tract damage. However, the radioprotective mechanism is unknown. In this study, we have investigated the expression of Hst-1/Fgf-4 in mouse small intestine after irradiation, and determined the role of HST-1/FGF-4 in mouse intestinal crypt cell survival and epithelial cell proliferation and restitution. We found the induction of endogenous Hst-1/Fgf-4 expression in intestine when mice are exposed to 9.0 Gy irradiation. Laser-captured microdissection (LCM) coupled with RT-PCR analysis revealed that expression of Hst-1/Fgf-4 was found in epithelial cell of the villi and crypt cells. Pretreatment of HST-1/FGF-4 caused an increase in the number of surviving crypt cells, and clearly suppresses the radiation-induced apoptosis of the crypt cells. Moreover, exogenous HST-1/FGF-4 enhances epithelial cell restitution and proliferation in an in vitro model. These data suggest that HST-1/FGF-4 is induced by irradiation injury, and that HST-1/FGF-4 will find a therapeutic role in the prevention of intestinal cell toxicity following intensive chemotherapy and radiation therapy protocols, and in allogenic cell transplantation.

HST-1/FGF-4 protects male germ cells from apoptosis under heat-stress condition.

Apoptosis plays an important role in controlling the number of male germ cells and eliminating defective germ cells during testicular development and spermatogenesis. We show here that fibroblast growth factor-4 (HST-1/FGF-4) may play a critical role as a survival factor for germ cells, protecting them from apoptosis. Testes of adult male mice that received an adenovirus carrying human HST-1/FGF-4 (AxHST-1) or a control adenovirus (AxCAwt) were exposed to mild hyperthermia, which causes germ cell apoptosis. An in situ terminal-deoxynucleotidyl transferase-mediated deoxy-UTP nick end-labeling (TUNEL) assay characterized germ cell apoptosis. The results indicated that HST-1/FGF-4 significantly reduced the apoptotic death of germ cells and prevented testicular weight loss and sperm count reduction. We also found that Hst-1/Fgf-4 present in testes is up-regulated in vivo when the testes are exposed to mild hyperthermia, and that endogenous Hst-1/Fgf-4 mRNA expression in Sertoli cells are also induced when the cells are exposed to mild hyperthermia in vitro. In addition, the MAPK cascade, which could increase an FGF-dependent survival signal, is activated by HST-1/FGF-4 stimuli in germ cells. On the other hand, upon HST-1/FGF-4 stimulation, lactate production from Sertoli cells were induced, which is indispensable nutrient for germ cell survival. These results suggest that HST-1/FGF-4 can act as an important physiological anti-apoptotic factor for male germ cells in stimulating lactate production of Sertoli cells upon heat stress, thereby promoting germ cell survival.

Differentiation of embryonic stem cells into hepatocytes: biological functions and therapeutic application.

Embryonic stem (ES) cells provide a unique source for tissue regeneration. We examined whether mouse ES cells can efficiently differentiate into transplantable hepatocytes. ES cells were implanted into mouse livers 24 hours after carbon tetrachloride intoxication; ES-derived cells with several hepatocyte-cell-markers were generated. They were able to grow in vitro and showed morphology consistent with typical mature hepatocytes and expressed hepatocyte-specific genes. After transplantation into the carbon tetrachloride-injured mouse liver, ES-derived green fluorescent protein-positive cells were incorporated into liver tissue and rescued mice from hepatic injury. No teratoma formation was observed in the transplant recipients. In conclusion, ES cells can provide a valuable tool for studying the molecular basis for differentiation of hepatocytes and form the basis for cell therapies.

Mammalian Rcd1 is a novel transcriptional cofactor that mediates retinoic acid-induced cell differentiation.

Rcd1, initially identified as a factor essential for the commitment to nitrogen starvation-invoked differentiation in fission yeast, is one of the most conserved proteins found across eukaryotes, and its mammalian homolog is expressed in a variety of differentiating tissues. Here we show that mammalian Rcd1 is a novel transcriptional cofactor and is critically involved in the commitment step in the retinoic acid-induced differentiation of F9 mouse teratocarcinoma cells, at least in part, via forming complexes with retinoic acid receptor and activation transcription factor-2 (ATF-2). In addition, antisense oligonucleotide treatment of embryonic mouse lung explants suggests that Rcd1 also plays a role in retinoic acid-controlled lung development.